Hydroxamic acid derivatives as antibacterials

ABSTRACT

A method for the treatment of bacterial infections in humans and non-human mammals, which comprises inhibiting bacterial growth by administering to a subject an antibacterially effective dose of a compound of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof:

BACKGROUND OF THE INVENTION

In general, bacterial pathogens are classified as either Gram-positiveor Gram-negative. Many antibacterial agents (including antibiotics) arespecific against one or other Gram-class of pathogens. Antibacterialagents effective against both Gram-positive and Gram-negative pathogensare therefore generally regarded as having broad spectrum activity.

Many classes of antibacterial agents are known, including thepenicillins and cephalosporins, tetracyclines, sulfonamides,monobactams, fluoroquinolones and quinolones, aminoglycosides,glycopeptides, macrolides, polymyxins, lincosamides, trimethoprim andchloramphenicol. The fundamental mechanisms of action of theseantibacterial classes vary.

Bacterial resistance to many known antibacterials is a growing problem.Accordingly there is a continuing need in the art for alternativeantibacterial agents, especially those which have mechanisms of actionfundamentally different from the known classes.

Amongst the Gram-positive pathogens, such as Staphylococci,Streptococci, Mycobacteria and Enterococci, resistant strains haveevolved/arisen which makes them particularly difficult to eradicate.Examples of such strains are methicillin resistant Staphylococcus aureus(MRSA), methicillin resistant coagulase negative Staphylococci (MRCNS),penicillin resistant Streptococcus pneumoniae and multiply resistantEnterococcus faecium.

Pathogenic bacteria are often resistant to the aminoglycoside, β-lactam(penicillins and cephalosporins), and chloramphenicol types ofantibiotic. This resistance involves the enzymatic inactivation of theantibiotic by hydrolysis or by formation of inactive derivatives. Theβ-lactam (penicillin and cephalosporin) family of antibiotics arecharacterised by the presence of a β-lactam ring structure. Resistanceto this family of antibiotics in clinical isolates is most commonly dueto the production of a “penicillinase” (β-lactamase) enzyme by theresistant bacterium which hydrolyses the β-lactam ring thus eliminatingits antibacterial activity.

Recently there has been an emergence of vancomycin-resistant strains ofenterococci (Woodford N. 1998 Glycopeptide-resistant enterococci: adecade of experience. Journal of Medical Microbiology. 47(10):849-62).Vancomycin-resistant enterococci are particularly hazardous in that theyare frequent causes of hospital based infections and are inherentlyresistant to most antibiotics. Vancomycin works by binding to theterminal D-Ala-D-Ala residues of the cell wall peptidioglycan precursor.The high-level resistance to vancomycin is known as VanA and isconferred by a genes located on a transposable element which alter theterminal residues to D-Ala-D-lac thus reducing the affinity forvancomycin.

In view of the rapid emergence of multidrug-resistant bacteria, thedevelopment of antibacterial agents with novel modes of action that areeffective against the growing number of resistant bacteria, particularlythe vancomycin resistant enterococci and β-lactam antibiotic-resistantbacteria, such as methicillin-resistant Staphylococcus aureus, is ofutmost importance.

Brief Description of the Invention

This invention is based on the finding that certain hydroxamic acidderivatives have antibacterial activity, and makes available a new classof antibacterial agents. The inventors have found that the compoundswith which this invention is concerned are antibacterial with respect toa range of Gram-positive and Gram-negative organisms.

Although it may be of interest to establish the mechanism of action ofthe compounds with which the invention is concerned, it is their abilityto inhibit bacterial growth which makes them useful. However, it ispresently believed that their antibacterial activity is due, at least inpart, to intracellular inhibition of bacterial polypeptide deformylase(PDF) enzyme.

Bacterial polypeptide deformylases (PDF) (EC 3.5.1.31), are a conservedfamily of metalloenzymes (Reviewed: Meinnel T, Lazennec C, Villoing S,Blanquet S, 1997, Journal of Molecular Biology 267, 749-761) which areessential for bacterial viability, their function being to remove theformyl group from the N-terminal methionine residue ofribosome-synthesised proteins in eubacteria. Mazel et al. (EMBO J.13(4):914-923, 1994) have recently cloned and characterised an E. coliPDF. As PDF is essential to the growth of bacteria and there is noeukaryotic counterpart to PDF, Mazel et al. (ibid), Rajagopalan et al.(J. Am. Chem. Soc. 119:12418-12419, 1997) and Becker et al., (J. BiolChem. 273(19):11413-11416, 1998) have each proposed that PDF is anexcellent anti-bacterial target.

The natural antibiotic actinonin (see for example J. C. S Perkin I,1975, 819) is a hydroxamic acid derivative of Structure (A):

In addition, various structural analogues of actinonin have also beenshown to have antibacterial activity (see for example Broughton et al.(Devlin et al. Journal of the Chemical Society. Perkin Transactions 1(9):830-841, 1975; Broughton et al. Journal of the Chemical Society.Perkin Transactions 1 (9):857-860, 1975).

Hydroxamic acid derivatives are also known in the field of matrixmetalloproteinase (MMP) inhibition. Many examples of the class have beensynthesised and their MMP inhibitory properties reported. A smallernumber have been reported to be active in animal models of diseasesmediated by MMPs, for example various cancers and rheumatoid arthritis.For reviews of the patent literature on hydroxamate MMP inhibitors, seefor example Beckett, Exp. Opin. Ther. Patents (1996) 6, 1305-1315, andBeckett & Whittaker, Exp. Opin. Ther. Patents (1998), 8(3), 259-282, andthe documents cited therein.

DESCRIPTION OF THE INVENTION

According to the present invention there is provided the use of acornpound of formula (I) or a pharmaceutically or veterinarilyacceptable salt thereof in the preparation of an antibacterialcomposition;

wherein:

R₁ represents hydrogen, or C₁-C₆ alkyl, C₁-C₆ alkyl substituted by oneor more halogen atoms, amino, hydroxy, or C₁-C₆ alkoxy;

R₂ represents a group R₁₀-(X)_(n-(ALK)) _(m- wherein)

-   -   R₁₀ represents hydrogen, or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, cycloalkyl, aryl, or heterocyclyl group, any of which        may be unsubstituted or substituted by (C₁-C₆)alkyl,        (C₁-C₆)alkoxy, hydroxy, mercapto, (C₁-C₆)alkylthio, amino, halo        (including fluoro, chloro, bromo and iodo), trifluoromethyl,        cyano, nitro, —COOH, —CONH₂-COOR^(A), -NHCOR^(A), -CONHR^(A),        -NHR^(A), -NR^(A)R^(B), or -CONR^(A)R^(B) wherein R^(A) and        R^(B) are independently a (C₁-C₆)alkyl group, and ALK represents        a straight or branched divalent C₁-C₆ alkylene, C₂-C₆        alkenylene, or C₂-C₆ alkynylene radical, and may be interrupted        by one or more non-adjacent —NH—, —O— or —S— linkages,    -   X represents —NH—, —O— or —S—, and    -   m and n are independently 0 or 1; and

A represents (i) a group of formula (IA), (IB), (IC) or (ID)

wherein:

-   -   R₃ represents hydrogen or C₁-C₆ alkyl and R₄ represents the side        chain of a natural or non-natural alpha amino acid or R₃ and R₄        when taken together with the nitrogen and carbon atoms to which        they are respectively attached form an optionally substituted        saturated heterocyclic ring of 5 to 8 atoms which ring is        optionally fused to a carbocyclic or second heterocyclic ring,    -   R₅ and R₆, independently represent hydrogen, or optionally        substituted C₁-C₈. alkyl, cycloalkyl, aryl, aryl(C₁-C₆ alkyl),        heterocyclic, or heterocyclic(C₁-C₆ alkyl), or R₅ and R₆ when        taken together with the nitrogen atom to which they are attached        form an optionally substituted saturated heterocyclic ring of 3        to 8 atoms which ring is optionally fused to a carbocyclic or        second heterocyclic ring, and    -   R₇ represents hydrogen, C₁-C₆ alkyl, or an acyl group.

PROVIDED THAT (a) R₅ and R₆ taken together with the nitrogen atom towhich they are attached do not form an optionally substituted saturatedheterocyclic ring of 3 to 8 atoms when R₁ and R₃ are hydrogen, R₂ ishydrogen, C₁-C₆ alkyl, phenyl, benzyl, 4-chlorophenylmethyl,4-nitrophenylmethyl, or 4-aminophenylmethyl and R₃ is hydrogen, methyl,isopropyl, isobutyl or benzyl; and (b) R₅ is not 2-pyridyl or2-thiazolyl when R₁ R₃ and R₆ are hydrogen, R₂ is n-pentyl and R₄ isisopropyl; and (c) R₅ and R₆ are not both ethyl when R₁ and R₃ arehydrogen, R₂ is n-pentyl and R₄ is methyl or isopropyl.

In another aspect, the invention provides a method for the treatment ofbacterial infections in humans and non-human mammals, which comprisesadministering to a subject suffering such infection an antibacteriallyeffective dose of a compound of formula (I) as defined above.

In a further aspect of the invention there is provided a method for thetreatment of bacterial contamination by applying an antibacteriallyeffective amount of a compound of formula (I) as defined above to thesite of contamination.

The compounds of formula (I) as defined above may be used ascomponent(s) of antibacterial cleaning or disinfecting materials.

According to a preferred embodiment, the various aspects of theinvention can be applied against vancomycin-, quinolone- and“β-lactam”-resistant bacteria and the infections they cause.

On the hypothesis that the compounds (I) act by inhibition ofintracellular PDF, the most potent antibacterial effect may be achievedby using compounds which efficiently pass through the bacterial cellwall. Thus, compounds which are highly active as inhibitors of PDF invitro and which penetrate bacterial cells are preferred for use inaccordance with the invention. It is to be expected that theantibacterial potency of compounds which are potent inhibitors of thePDF enzyme in vitro, but are poorly cell penetrant, may be improved bytheir use in the form of a prodrug, ie a structurally modified analoguewhich is converted to the parent molecule of formula (I), for example byenzymic action, after it has passed through the bacterial cell wall.

As used herein the term “(C₁-C₆)alkyl” means a straight or branchedchain alkyl moiety having from 1 to 6 carbon atoms, including forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term “divalent (C₁-C₆)alkylene radical” means asaturated hydrocarbon chain having from 1 to 6 carbon atoms and twounsatisfied valencies.

As used herein the term “(C₂-C₆)alkenyl” means a straight or branchedchain alkenyl moiety having from 2 to 6 carbon atoms having at least onedouble bond of either E or Z stereochemistry where applicable. The termincludes, for example, vinyl, allyl, 1- and 2-butenyl and2-methyl-2-propenyl.

As used herein the term “divalent (C₂-C₆)alkenylene radical” means ahydrocarbon chain having from 2 to 6 carbon atoms, at least one doublebond, and two unsatisfied valencies.

As used herein the term “C₂-C₆ alkynyl” refers to straight chain orbranched chain hydrocarbon groups having from two to six carbon atomsand having in addition one triple bond. This term would include forexample, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and5-hexynyl.

As used herein the term “divalent (C₂-C₆)alkynylene radical” means ahydrocarbon chain having from 2 to 6 carbon atoms, at least one triplebond, and two unsatisfied valencies.

As used herein the term “cycloalkyl” means a-saturated alicyclic moietyhaving from 3-8 carbon atoms and includes, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the term “cycloalkenyl” means an unsaturated alicyclicmoiety having from 3-8 carbon atoms and includes, for example,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyland cyclooctenyl. In the case of cycloalkenyl rings of from 5-8 carbonatoms, the ring may contain more than one double bond.

As used herein the term “aryl” refers to a mono-, bi- or tri-cycliccarbocyclic aromatic group, and to groups consisting of two covalentlylinked monocyclic carbocyclic aromatic groups. Illustrative of suchgroups are phenyl, biphenyl and napthyl.

As used herein the term “heteroaryl” refers to a 5- or6- memberedaromatic ring containing one or more heteroatoms, and optionally fusedto a benzyl or pyridyl ring; and to groups consisting of two covalentlylinked 5- or 6- membered aromatic rings each containing one or moreheteroatoms; and to groups consisting of a monocyclic carbocyclicaromatic group covalently linked to a 5- or 6- membered aromatic ringscontaining one or more heteroatoms;. Illustrative of such groups arethienyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl,pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl,oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,4-([1,2,3]-thiadiazoly-4-yl)phenyl and 5-isoxazol-3-ylthienyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic”includes “heteroaryl” as defined above, and in particular means a 5-7membered aromatic or non-aromatic heterocyclic ring containing one ormore heteroatoms selected from S, N and O, and optionally fused to abenzene ring, including for example, pyrrolyl, furyl, thienyl,piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl,pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl,benzimidazolyl, maleimido, succinimido, phthalimido and1,3-dioxo-1,3-dihydro-isoindol-2-yJ groups.

As used herein the term “acyl” means a group R₂₀C(O)- where R₂₀ is(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₃-C₇)cycloalkyl, phenyl, heterocyclyl,phenyl(C₁-C₆)alkyl, heterocyclyl(C₁-C₆)alkyl,(C₃-C₇)cycloalkyl(C₁-C₆)alkyl, phenyl(C₂-C₆)alkenyl,heterocyclyl(C₂-C₆)alkenyl, (C₃-C₇)cycloalkyl(C₂-C₆)alkenyl, any ofwhich R₂₀ groups may be substituted.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with upto four substituents, each of which independently may be (C₁-C₆)alkyl,benzyl, (C₁-C₆)alkoxy, phenoxy, hydroxy, mercapto, (C₁-C₆)alkylthio,amino, halo (including fluoro, chloro, bromo and iodo), trifluoromethyl,nitro, —COOH, —CONH₂, -COR^(A), -COOR^(A), -NHCOR^(A), -CONHR^(A),-NHR^(A), -NR^(A)R^(B), or -CONR^(A)R^(B) wherein R^(A) and R^(B) areindependently a (C₁-C₆)alkyl group. In the case where “substituted”means benzyl, the phenyl ring thereof may itself be substituted with anyof the foregoing, except benzyl.

As used herein the terms “side chain of a natural alpha-amino acid” and“side chain of a non-natural alpha-amino acid” mean the group R^(x) inrespectively a natural and non-natural amino acid of formulaNH₂—CH(R^(x))—COOH.

Examples of side chains of natural alpha amino acids include those ofalanine, arginine, asparagine, aspartic acid, cysteine, cystine,glutamic acid, histidine, 5-hydroxylysine, 4-hydroxyproline, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, α-aminoadipic acid, α-amino-n-butyricacid, 3,4-dihydroxyphenylalanine, homoserine, α-methylserine, ornithine,pipecolic acid, and thyroxine.

In natural alpha-amino acid side chains which contain functionalsubstituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl,imidazolyl, or indolyl groups as in arginine, lysine, glutamic acid,aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, andcysteine, such functional substituents may optionally be protected.

Likewise, in the side chains of non-natural alpha amino acids whichcontain functional substituents, for example amino, carboxyl, hydroxy,mercapto, guanidyl, imidazolyl, or indolyl groups, such functionalsubstituents may optionally be protected.

The term “protected” when used in relation to a functional substituentin a side chain of a natural or non-natural alpha-amino acid means aderivative of such a substituent which is substantially non-functional.The widely used handbook by T. W. Greene and P. G. Wuts “ProtectiveGroups in Organic Synthesis” Second Edition, Wiley, New York, 1991reviews the subject. For example, carboxyl groups may be esterified (forexample as a C₁-C₆ alkyl ester), amind groups may be converted to amides(for-example as a NHCOC₁-C₆ alkyl amide) or carbamates (for example asan NHC(=O)OC₁-C₆ alkyl or NHC(=O)OCH₂Ph carbamate), hydroxyl groups maybe converted to ethers (for example an OC₁-C₆ alkyl or a O(C₁-C₆alkyl)phenyl ether) or esters (for example a OC(=O)C₁-C₆ alkyl ester)and thiol groups may be converted to thioethers (for example atert-butyl or benzyl thioether) or thioesters (for example a SC(=O)C₁-C₆alkyl thioester).

Salts of the compounds of the invention include physiologicallyacceptable acid addition salts for example hydrochlorides,hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates,phosphates, acetates, citrates, succinates, lactates, tartrates,fumarates and maleates. Salts may also be formed with bases, for examplesodium, potassium, magnesium, and calcium salts.

There are several actual or potential chiral centres in the compoundsaccording to the invention because of the presence of asymmetric carbonatoms. The presence of several asymmetric carbon atoms gives rise to anumber of diastereoisomers with R or S stereochemistry at each chiralcentre. The invention includes all such diastereoisomers and mixturesthereof. Currently, the preferred stereoconfiguration of the carbon atomcarrying the R₂ group is R; that of the carbon atom carrying the R₄group (when asymmetric) is S; and that of the carbon atom carrying theR₁ group (when asymmetric) is R.

In the compounds for use according to the invention and in the novelcompounds of the invention:

R₁ may be, for example, hydrogen, hydroxy, methoxy, methyl, ortrifuoromethyl.

Hydrogen is currently preferred.

R₂ may be, for example:

-   -   optionally substituted C₁-C₈ alkyl, C₃-C₆ alkenyl, C₃-C₆ alkynyl        or cycloalkyl;    -   phenyl(C₁-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆        alkynyl)-optionally substituted in the phenyl ring;    -   cycloalkyl(C₁-C₆ alkyl)-, cycloalkyl(C₃-C₆ alkenyl)- or        cycloalkyl(C₃-C₆ alkynyl) -optionally substituted in the        cycloalkyl ring;    -   heterocyclyl(C₁-C₆ alkyl)-, heterocyclyl(C₃-C₆ alkenyl)- or        heterocyclyl(C₃-C₆ alkynyl)- optionally substituted in the        heterocyclyl ring; or    -   CH₃(CH₂)_(p)O(CH₂)_(q) or CH₃(CH₂)_(p)S(CH₂)_(q)-, wherein p is        0, 1, 2 or 3 and q is 1, 2or 3.

Specific examples of R₂ groups include

-   -   methyl, ethyl, n- and iso-propyl, n- and iso-butyl, n-pentyl,        iso-pentyl 3-methyl-but -1-yl, n-hexyl, n-heptyl, n-acetyl,        n-octyl, methylsulfanylethyl, ethylsulfanylmethyl,        2-methoxyethyl, 2-ethoxyethyl, 2-ethoxymethyl, 3 hydroxypropyl,        allyl, 3-phenylprop-3-en-1-yl, prop-2-yn-1-yl, 3-phenylprop-2-yn        -1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yl, but-2-yn-1-yl,        cyclopentyl, cyclohexyl, cyclopentylmethyl, cyclopentylethyl,        cyclopentylpropyl, cyclohexylmethyl, cyclohexylethyl,        cyclohexylpropyl, furan-2-ylmethyl, furan-3-methyl, methyl,        tetrahydrofuran-2-ylmethyl, tetra hydrofuran-2-ylmethyl,        piperidinylmethyl, phenylpropyl, 4-chlorophenylpropyl,        4-methylphenylpropyl, 4-methoxyphenylpropyl, benzyl,        4-chlorobenzyl, 4-methylbenzyl, and 4-methoxybenzyl.    -   Presently preferred groups at R₂ are n-propyl, n-butyl,        n-pentyl, benzyl and cyclopentylmethyl.

R₃ may be, for example, hydrogen or methyl, with hydrogen presentlypreferred.

R₄ may be, for example

-   -   the characterising group of a natural a-amino acid, for example        isopropyl, benzyl, or 4-hydroxyphenylmethyl, in which any        functional group may be protected, any amino group may be        acylated and any carboxyl group present may be amidated; or    -   a group -[Alk]_(n)R₉ where Alk is a (C₁-C₆)alkylene or        (C₂-C₆)alkenylene group optionally interrupted by one or more        —O—, or —S— atoms or —N(R₁₂)- groups [where R₁₂ is a hydrogen        atom or a (C₁-C₆)alkyl group], n is 0 or 1, and R₉ is hydrogen        or an optionally substituted phenyl, aryl, heterocyclyl,        cycloalkyl or cycloalkenyl group or (only when n is 1) R₉ may        additionally be hydroxy, mercapto, (C₁-C₆)alkylthio, amino,        halo, trifluoromethyl, nitro, —COOH, —CONH₂ -COOR^(A),        -NHCOR^(A), -CONHR^(A), -NHR^(A), -NR^(A)R^(B), or        -CONR^(A)R^(B) wherein R^(A) and R^(B) are independently a        (C₁-C₆)alkyl group; or    -   a benzyl group substituted in the phenyl ring by a group of        formula -OCH₂COR₈ where R_(B) is hydroxyl, amino, (C₁-C₆)alkoxy,        phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino, di((C₁-C₆)alkyl)amino,        phenyl(C₁-C₆)alkylamino; or    -   a heterocyclic(C₁-C₆)alkyl group, either being unsubstituted or        mono- or di-substituted in the heterocyclic ring with halo,        nitro, carboxy, (C₁-C₆)alkoxy, cyano, (C₁-C₆)alkanoyl,        trifluoromethyl (C₁-C₆)alkyl, hydroxy, formyl, amino,        (C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino, mercapto,        (C₁-C₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁-C6)alkyl or        (C₁-C₆)alkylphenylmethyl; or    -   a group -CR_(a)R_(b)R_(C) in which:        -   each of R_(a) R_(b) and R_(c) is independently hydrogen,            (C₁-C₆)alkyl, (C₂-C₆) alkenyl, (C₂-C₆)alkynyl,            phenyl(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl; or        -   R_(c) is hydrogen and R_(a) and R_(b) are independently            phenyl or heteroaryl such as pyridyl; or        -   R_(c) is hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,            (C₂-C₆)alkynyl, phenyl(C₁-C₆) alkyl, or (C₃-C₈)cycloalkyl,            and R_(a) and R_(b) together with the carbon atom to which            they are attached form a 3- to 8- membered cycloalkyl or a            5- to 6-membered heterocyclic ring; or        -   R_(a), R_(b) and R_(c) together with the carbon atom to            which they are attached form a tricyclic ring (for example            adamantyl); or        -   R_(a) and R_(b) are each independently (C₁-C₆)alkyl,            (C₂-C₆)alkenyl, (C₂-C₆) alkynyl, phenyl(C₁-C₆)alkyl, or a            group as defined for R_(c) below other than hydrogen, or            R_(a) and R_(b) together with the carbon atom to which they            are attached form a cycloalkyl or heterocyclic ring, and            R_(c) is hydrogen, —OH, —SH, halogen, —CN, —CO₂H,            (C₁-C₄)perfluoroalkyl, -CH₂OH, —CO₂(C₁-C₆)alkyl,            —O(C₁-C₆)alkyl, —O(C₂-C₆)alkenyl, —S(C₁-C₆) alkyl,            —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆) alkyl, —S(C₂-C₆)alkenyl,            —SO(C₂-C₆) alkenyl, —SO₂(C₂-C₆)alkenyl or a group -Q-W            wherein Q represents a bond or —O—, —S—, —SO—or —SO₂- and W            represents a phenyl, phenylalkyl, (C₃-C₈)cycloalkyl,            (C₃-C₈)cycloalkylalkyl, (C₄-C₈) cycloalkenyl,            (C₄-C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl            group, which group W may optionally be substituted by one or            more substituents independently selected from, hydroxyl,            halogen, —CN, —CO₂H, —CO₂(C₁-C₆)alkyl, —CONH₂,            —CONH(C₁-C₆)alkyl, —CONH(C₁-C₆alkyl)₂, —CHO, —CH₂OH,            (C₁-C₄)perfluoroalkyl, —O(C₁-C₆)alkyl, —S(C₁-C₆) alkyl,            —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)alkyl, —NO₂, —NH₂, —NH(C₁-C₆)            alkyl, —N((C₁-C₆)alkyl)₂, —NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl,            (C₂-C₆) alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl,            (C₄-C₈)cycloalkenyl, phenyl or benzyl.    -   Examples of particular R₄ groups include methyl, ethyl,        isopropyl, benzyl, 4-chlorobenzyl, 4-hydroxybenzyl, phenyl,        cyclohexyl, cyclohexylmethyl, pyridin-3-ylmethyl,        tert-butoxymethyl, naphthylmethyl, iso-butyl, sec-butyl,        tert-butyl, 1-benzylthio-1-methylethyl,        1-methylthio-1-methylethyl, 1-mercapto-1-methylethyl,        1-methoxy-1-methylethyl, 1-hydroxy-1-methylethyl,        1-fluoro-1-methylethyl, 4,4-dimethyl-prop-1-en-4-yl,        4,4-dimethyl-prop-4-yl hydroxymethyl, 2-hydroxethyl,        2-carboxyethyl, 2-methylcarbamoylethyl, 2-carbamoylethyl, and        4-aminobutyl. Presently preferred R₄ groups include tert-butyl,        iso-butyl, benzyl and methyl.

R₃ and R₄ when taken together with the nitrogen and carbon atoms towhich they are respectively attached may form an optionally substitutedsaturated heterocyclic ring of 5 to 8 atoms. For example, R₃ and R₄ mayform a bridge between the nitrogen and carbon atoms to which they areattached, said bridge being represented by the divalent radical—(CH₂)₃₋₆—, or —(CH₂),—O—(CH₂)_(s)—, or —(CH₂)_(r)—S—(CH₂)_(s)—, whereinr and s are each independently 1, 2 or 3 with the proviso that r+s =2,3, 4, or 5.

R₅ and R₆ may independently be, for example, hydrogen, methyl, ethyl,tert-butyl, n-heptyl, cyclopentyl, cyclohexyl,phenyl,2-ethoxycarbonyl-eth-2-yl, pyrid-2-yl, 1,1,3,3-tetramethylbutyl,benzyl, 2,6-dimethyl-4-tert-butyl-phenyl,diphenylmethyl,4-chlorophenyl-phenylmethyl, 2-fluorophenyl-phenylmethyl,1-(4-fluorophenyl)-l-phenyl-1-amino-methyl, 1,1-diphenylprop-3-yl,3-phenyl-thiazolyl, or 2-hydroxyethyl; or R₅ and R₆ when taken togetherwith the nitrogen atom to which they are attached may form a saturated5- to 8-membered monocyclic N-heterocyclic ring which is attached viathe N atom and which optionally contains -N(R₁₁)- wherein R₁₁ ishydrogen or C₁-C₆ alkyl, benzyl, acyl, or an amino protecting group, O,S SO or SO₂ as a ring member, and/or is optionally substituted on one ormore C atoms by hydroxy, C₁-C₆ alkyl, hydroxy(C₁-C₆ alkyl)-, C,-C6alkoxy, oxo, ketalised oxo, amino, mono(C₁-C₆ alkyl)amino, di(C₁-C₆alkyl)amino, carboxy, C₁-C₆ alkoxycarbonyl, hydroxymethyl, C₁-C6alkoxymethyl, carbamoyl, mono(C₁-C₆ alkyl)carbamoyl, di(C₁-C₆ alkyl)carbamoyl, or hydroxyimino.

-   -   Examples of such rings are substituted or unsubstituted        1-pyrrolidinyl, piperidin-1-yl, 1-piperazinyl,        hexahydro-1-pyridazinyl, morpholin-4-yl,        tetrahydro-1,4-thiazin4-yt, tetrahydro-1,4-thiazin4-yl 1-oxide,        tetrahydro-1,4-thiazin-4-yl 1, 1-dioxide, hexahydroazipino, or        octahydroazocino. Substituted examples of the foregoing are        2-(methylcarbamoyl)-1-pyrrolidinyl, 2-(hydroxymethyl)        -1-pyrrolidinyl, 4-hydroxypiperidino, 2-(methylcarbamoyl)        piperidino, 4-hydroxyiminopiperidino, 4-methoxypiperidino,        4-methylpiperidin-1yl,4-benzylpiperidin-1-yl,4-acetylpiperidin-1-yl,4-methyl-1-piperazinyl,        4-phenyl-1-piperazinyl, 1 ,4-dioxa-8-azaspiro[4, 5]decan-8yl,        hexahydro-3-(methylcarbamoyl)-2-pyridazinyl, and        hexahydro-1-(benzyloxycarbonyl) -2-pyridazinyl,        decahydroisoquinolin-2-yl, and 1,2,3,4-tetrahydroisoquinolin        -2-yl.

When A is a group of formula (IA), it is currently preferred that R₅ bemethyl or hydrogen, and R₆ be methyl.

R₇ may be, for example, hydrogen, or a group R₂₀C(O)- where R₂₀ is a(C₁-C₆)alkyl group such as methyl or ethyl.

A specific example of a compound having PDF inhibiting and antibacterialactivity in accordance with the invention is:

-   -   N¹-(1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl)-N⁴-hydroxy-2        R-butyl-succinamide        and pharmaceutically acceptable salts, hydrates and solvates        thereof.

Compounds for use in accordance with the invention may be prepared bymethods described in the literature for the preparation of hydroxamateMMP inhibitors, for example the patent publications relating to suchcompounds cited in Beckett, Exp. Opin. Ther. Patents (1996) 6,1305-1315, and Beckeff & Whittaker, Exp. Opin. Ther. Patents (1998),8(3), 259-282.

Compositions with which the invention is concerned may be prepared foradministration by any route consistent with the pharmacokineticproperties of the active ingredient(s).

Orally administrable compositions may be in the form of tablets,capsules, powders, granules, lozenges, liquid or gel preparations, suchas oral, topical, or sterile parenteral solutions or suspensions.Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

For topical application to the skin, the active ingredient(s) may bemade up into a cream, lotion or ointment. Cream or ointment formulationswhich may be used for the drug are conventional formulations well knownin the art, for example as described in standard textbooks ofpharmaceutics such as the British Pharmacopoeia.

The active ingredient(s) may also be administered parenterally in asterile medium. Depending on the vehicle and concentration used, thedrug can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as a local anaesthetic, preservative andbuffering agents can be dissolved in the vehicle.

Safe and effective dosages for different classes of patient and fordifferent disease states will be determined by clinical trial as isrequired in the art. It will be understood that the specific dose levelfor any particular patient will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

The following examples are of compounds of formula (I) above having PDFinhibiting activity and antibacterial activity in accordance with theinvention

¹H and ¹³C NMR spectra were recorded using a Bruker AC 250E spectrometerat 250.1 and 62.9 MHz, respectively. Elemental microanalyses wereperformed by MEDAC Ltd. Department of Chemistry, Brunel University,Uxbridge, Middlesex UB8 3PH. L-Tert-leucine-N-methylamide was preparedaccording to established literature methods.

EXAMPLE 1

N¹-(1S-Methylcarbamoyl-2-phenyl-ethyl)-N⁴-hydroxy-2 R-propyl-succinamide

The title compound was prepared by the method described in WO 92/13831(Example 1), substituting valeryl chloride for 4-methylvaleryl chloride.

m.p. =191- 193° C. ¹H-NMR δ (CD₃OD, partial D exchange); 8.12 (0.5H, d,J=8.1 Hz), 7.96-7.87 (5H, m), 4.55-4.40 (1H, m), 3.11 (1 H, dd, J=6.4,13.7 Hz), 2.89 (1H, dd, J=8.9, 13.7 Hz), 2.65, 2.63 (3H, 2s), 2.60-2.50(1H, m), 2.20 (1H, dd, J=8.0, 14.6 Hz), 2.06 (1H, dd, J=6.7, 14.6 Hz),1.48-1.00 (4H, m) and 0.78 (3H, t, J=7.1 Hz). ¹³C-NMR δ (CD₃OD); 177.0,174.0, 170.8, 138.8, 130.3, 129.4, 127.7, 56.3, 44.4, 38.7, 36.4, 35.6,26.3 21.3 and 14.3. IR (KBr, v_(max) cm⁻¹); 3292, 2957, 1637, 1560 and1541. Found: C 60.18, H 7.45, N 12.52%; C₁₇H₂₅, N₃O₄0.2H₂O requires C60.23, H 7.55, N 12.39%.

The compounds of Examples 2 and 3 were prepared by analogy with Example1.

EXAMPLE 2

N-¹-(1S-Methylcarbamoyl-2,2-dimethyl-propyl)-N⁴-hydroxy-2R-propyl-succinamide

m.p. 200° C. ¹H-NMR (CD₃OD); 4.19 (1H, s), 2.90-2.76 (1H, m), 2.68 (3H,s), 2.31 (1H, dd, J=7.9, 14.6 Hz), 2.15 (1H, dd, J=6.5, 14.6 Hz),1.60-1.40 (1H, m), 1.40-1.14 (3H, m), 0.95 (9H, s) and 0.85 (3H, t,J=7.0 Hz). ¹³C-NMR (CD₃OD); 176.9, 173.3, 170.7, 62.2, 43.6, 36.5, 35.7,35.3, 27.17, 26.0, 21.4 and 14.4. IR (KBr, v_(max) cm⁻¹); 1682, 1634,1544, 1470, 1413, 1369 and 1248. Found C 55.35H 8.84 N 13.92%;C₁₄H₂₇N₃O₄ requires C 55.79, H 9.03, N 13.94%.

EXAMPLE 3

N¹-(1S-Dimethylcarbamoyl-2,2-dimethyl-l1-propyl)-N⁴-hydroxy-2R-butyl-succinamide

Off white solid. m.p. 165-166° C. ¹H-NMR; δ (CDCl₃), 4.87 (1H, s), 3.19(3H, s), 2.93 (3H, s), 2.83 (1H, m), 2.35 (1H, dd, J=7.8, 14.6Hz), 2.19(1H, dd, J=6.3, 14.5 Hz), 1.59-1.06 (6H, br m), 1.01 (9H, s) and 0.87(3H, t, J=6.9 Hz). ¹H-NMR; δ (CDCl₃), 177.5, 173.6, 171.1, 71.1, 56.6,42.3, 39.2, 36.6, 36.4, 33.6, 30.8, 27.5, 24.0 and 14.7. LRMS: +ve ion352 [M+Na], -ve ion 328 [M-H].

EXAMPLE 4

By methods described in the literature analogous to those used forExample 1-3 above, the following compounds of formula (I) above, whereinA is a group of formula (IA) were prepared: R₁ R₂ R₃ R₄ R₅ R₆ Hcyclopentylmethyl H benzyl H methyl H iso-butyl H iso-propyl H methyl Hiso-butyl H benzyl H phenyl H iso-butyl H tert-butyl H phenyl Hiso-butyl H 1-methyl-1-methylthio- H methyl ethyl H HO(CH₂)₁₄— Htert-butyl H methyl H cyclopentylmethyl H tert-butyl H tert-butyl Hcyclopentylmethyl H 4,4-dimethyl-prop-1-en- H cyclohexyl 4-yl Hcyclopentylmethyl H 4,4-dimethyl-prop-4-yl H cyclohexyl H iso-butyl Htert-butyl H methyl H iso-butyl H iso-butyl H 2-ethoxycarbonyl-eth- 2-ylH iso-butyl H tert-butyl H tert-butyl H iso-butyl H tert-butyl methylmethyl H iso-butyl H tert-butyl H pyrid-2-yl H cyclopentylmethyl Htert-butyl H benzyl OH iso-butyl H tert-butyl H 2,6-dimethyl-4-tert-butyl-phenyl OH iso-butyl H tert-butyl H diphenylmethyl OH iso-butyl Htert-butyl H 4-chlorophenyl- phenylmethyl OH iso-butyl H tert-butyl H1,1-diphenylprop-3-yl OH iso-butyl H tert-butyl H 3-phenyl-thiazolyl OHiso-butyl H tert-butyl H 1-(4-fluorophenyl)-1- phenyl-1-amino-methyl OHiso-butyl H tert-butyl H 2-fluorophenyl- phenylmethyl

EXAMPLE 5

N¹-(1R,S-tert-Butylcarbamoyl-2,2-dimethyl-1-propyl)-N⁴-hydroxy-2R-butyl-succinamide

The title compound was prepared by Ugi reaction of the appropriatehomochiral succinate ester with trimethylacetaldehyde and ammonia,followed by conversion to the desired hydroxamic acid, as described inGB-2298423-A. The starting succinates were prepared by the methoddescribed in WO 92/13831.

¹H-NMR: δ (CD₃OD; mixture of diastereoisomers); 7.39 (0.5H, s), 7.32(0.5H, s), 4.08 (0.5H, s) 4.02 (0.5H, 2s), 2.80-2.70 (1H, m), 2.32-2.19(1H, m), 2.18-2.00 (1H, m), 1.60-1.04 (6H, m), 1.22 (4.5H, s), 1.21(4.5H, s), 0.90 (4.5H, s), 0.87 (4.5H, s) and 0.85-0.76 (3H, m). ¹³C-NMR(CD₃OD); 177.0, 176.8, 171.8, 170.9, 62.8, 62.2, 52.2, 52.1, 43.8, 43.7,36.6, 36.4, 35.8, 35.4, 35.3, 35.1, 28.9, 28.8, 27.4, 27.2, 21.5, 21.4,14.3 and 14.3. IR (KBr, v_(max) cm-⁻¹); 3313, 2963, 1637, 1546, 1456,1395, 1364, 1264, 1225 and 1188.

EXAMPLE 6

6-Biphenyl-4-yl-3R-(piperidine-1-carbonyl)-hexanoic acid hydroxyamide

The title compound was prepared by a method analogous to that used forthe preparation of compounds of Examples 1-4, except that piperidine wasused in the coupling reaction in place of the amino acid derivative.

m.p. 149-150° C. ¹H-NMR: δ((CD₃)₂SO), 10.37 (1H, s), 8.69 (1H, s),7.69-7.19 (9H, m), 3.59-3.24 (4H, m), 3.24-3.08 (1H, m), 2.65-2.43 (2H,m), 2.25 (1H, dd, J =7.8, 14.6 Hz), 2.01 (1H, dd, J=6.0, 14.7 Hz) and1.64-1.20 (10H, m). ¹³C-NMR; δ((CD₃)₂SO), 172.3, 168.0, 141.6, 140.5,138.0, 129.3, 127.5, 126.9, 126.8, 46.4, 42.6-6, 36.3, 35.4, 35.1, 31.8,28.5, 26.5, 25.8 and 24.5. IR (reflection disc, v_(max), cm⁻¹); 3230,2939, 2855, 1659, 1612, 1461.

BIOLOGICAL EXAMPLE A

i) Cloning of the Escherichia coli PDF gene.

The E. coli PDF gene was cloned in pET24a(+) (designated. pET24-PDF) andwas used to transform BL21 DE3 cells from Novagen Inc, (Madison,Wisconsin). Clones were selected at 37° C. on YT agar plates (8g/ltyptone, 5g/yeast extract, NaCl 5g/l, agar 15g/l) supplemented with30μg/ml kanamycin.

ii) Expression of PDF

A 20 ml overnight culture of BL21 DE3 cells harbouring pET24-PDF wasused to infect 500 ml 2×YT broth (16 g/l typtone, 10 g/l yeast extract,NaCl 5g/l) containing 30 ug/ml kanamycin in a 2 liter baffled flask andgrown at 37° C. with shaking to an OD₆₀₀ 0.6. The culture was theninduced by adjusting the medium to 1.0 mM isopropyl β-Dthiogalactopyranoside (IPTG). The induction was allowed to proceed for afurther 3 hours at 37° C., the cells were harvested by centrifugationand the cell pellet washed with 250 ml phosphate buffered saline (PBS)and the pellet stored at −70° C.

iii) Preparation of Soluble Protein Fraction.

The cells from a 1 liter expression were resuspeneded in 2×25 ml of icecold phosphate buffered saline. The cell suspension was sonicated on iceusing an MSE Soniprep 150 fitted with a medium probe and at an amplitudeof 20-25 microns in 6×20 second pluses. The resulting suspension wasthen cleared by centrifugation at 20,000 ×g for 15 minutes. Thesupematant was then used for further purification of the enzyme.

iv) PDF Purification

E coli lysate from a 11 culture in phosphate buffered saline (PBS) wereadjusted to 2M ammonium sulphate. A 15 ml phenyl sepharose column wasequilibrated with PBS/2M ammonium sulphate at 4° C. The lysate wasloaded on the column and washed with equilibration buffer. The columnwas eluted by reducing the ammonium sulphate concentration from 2M to 0Mover 10 column volumes. 5ml fractions were collected and analysed bySDS-PAGE. The fractions containing the majority of the 20 kDa PDF werepooled. The pooled fractions were concentrated using a 3 kDa cutoffmembrane to a volume of 5ml. The fraction was then loaded onto aSuperdex 75 (size exclusion chromatography) column equilibrated in PBS.The concentrated PDF pool eluted at one ml/min at 4° C. and 5mifractions collected and analysed by SDS-PAGE. The purest fractions werepooled and stored at −70° C.

(v) PDF in vitro assay

The assay was performed in a single 96 well plate in a final volume of100 μl containing:

-   -   20 μl PDF (4 μg/ml)    -   20 μl 100mM Hepes pH 7.0+1M KCI +0.05% Brij    -   10 μl serial dilution of test compound in 20% DMSO    -   50 μl formyl-Met-Ala-Ser (8 mM)

The assay was incubated at 37° C. for 30 minutes. The free amino groupof the deformylated (Met-Ala-Ser) product was detected usingfluorescamine, by the following additions:

-   -   50 μl 0.2 M borate pH 9.5    -   50 μl fluorescamine (150 μg/ml in dry dioxane)

Fluorescence was quantified on SLT Fluostar plate reader using anexcitation wavelength of 390nM and an emission wavelength of 485nM.Standard control reactions are a no inhibitor reaction which providesthe zero inhibition figure and a no enzyme and no inhibitor reactionwhich provides the 100% inhibition figure. The data was analysed byconversion of the fluorescence units to % inhibition and the inhibitorconcentration plotted against % inhibition. The data was fitted to asigmoidal function: y =A+((B−A)/(1+((C/x)^(D)))), wherein A representszero-inhibition, B represents 100% inhibition and C represents the IC₅₀,D represents the slope. The IC₅₀ represents the concentration ofinhibitor (nM) required to decrease enzyme activity by 50%.

The test compounds were found to inhibit bacterial PDF in vitro.

BIOLOGICAL EXAMPLE B

Minimal inhibitory concentrations (MIC) of the test compounds against E.coli strain DH5β (Genotype; F-φ80d/acZΔM15Δ(/acZYA-argF)U169 deoR recA1endA1 hsdR17(r_(k) ⁻, m_(k) ⁺)phoA supE44λ⁻thi-1 gyrA96 re/A1) obtainedfrom GibcoBRL Life Technologies, were determined as follows. Stocksolutions of test compound were prepared by dissolution of each compoundin dimethylsulfoxide at 10 mM. For the determination of the minimalinhibitory concentration, two fold serial dilutions were prepared in2×YT broth (typtone 16 g/l, yeast extract 10 g/l, sodium chloride 5 globtained from BIO 101 Inc, 1070 Joshua Way, Vista, CA92083, USA) toyield 0.05 ml compound-containing medium per well. Inocula were preparedfrom cultures grown overnight in 2×YT broth at 37° C. Cell densitieswere adjusted to absorbance at 660 nm (A₆₆₀)=0.1; the opticaldensity-standardized preparations were diluted 1:1000 in 2×YT broth; andeach well inoculated with 0.05 ml of the diluted bacteria. Microtiterplates were incubated at 37° C. for 18 hours in a humidified incubator.The test compounds had MIC's of 200 μM or less against one or both ofthe test organisms.

1. A method for the treatment of bacterial infections in humans andnon-human mammals, which comprises inhibiting bacterial growth byadministering to a subject an antibacterially effective dose of acompound of formula (I) or a pharmaceutically or veterinarily acceptablesalt thereof:

wherein: R₁ represents hydrogen, or C₁-C₆ alkyl, C₁-C₆ alkyl substitutedby one or more halogen atoms, amino, hydroxy, or C₁-C₆ alkoxy; R₂represents a group R₁₀-(X)_(n)-(ALK)_(m)- wherein R₁₀ representshydrogen, or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, cycloalkyl,aryl, or heterocyclyl group, any of which may be unsubstituted orsubstituted by C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxy, mercapto, C₁-C₆alkylthio, amino, halo, trifluoromethyl, cyano, nitro, —COOH,—CONH₂,-COOR^(A),-NHCOR^(A),-CONHR^(A),-NHR^(A), -NR^(A)R^(B), or-CONR^(A)R^(B) wherein R^(A) and R^(B) are independently a (C₁-C₆) alkylgroup, and ALK represents a straight or branched divalent C₁-C₆alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene radical, and may beinterrupted by one or more non-adjacent —NH—, —O— or —S— linkages, Xrepresents —NH—, —O—, or —S—, and m and n are independently 0 or 1; andA represents (i) a group of formula (IA), (IB), (IC) or (ID)

wherein: R₃ represents hydrogen or C₁-C₆ alkyl and R₄ represents theside chain of a natural or non-natural alpha amino acid or R₃ and R₄when taken together with the nitrogen and carbon atoms to which they arerespectively attached form an optionally substituted saturatedheterocyclic ring of 5 to 8 atoms which ring is optionally fused to acarbocyclic or second heterocyclic ring, PROVIDED THAT the fragment offormula

in (IA) is not a fragment of formula

R₅ and R₆, independently represent hydrogen, or optionally substitutedC₁-C₈ alkyl, cycloalkyl, aryl, aryl (C₁-C₆ alkyl), heterocyclic, orheterocyclic (C₁-C₆ alkyl), or R₅ and R₆ when taken together with thenitrogen atom to which they are attached form an optionally substitutedsaturated heterocyclic ring of 3 to 8 atoms which ring is optionallyfused to a carbocyclic or second heterocyclic ring, and R₇ representshydrogen, C₁-C₆ alkyl, or an acyl group. PROVIDED THAT (a) R₅ and R₆taken together with the nitrogen atom to which they are attached do notform an optionally substituted saturated heterocyclic ring of 3 to 8atoms when R₁ and R₃ are hydrogen, R₂ is hydrogen, C₁-C₆ alkyl, phenyl,benzyl, 4-chlorophenylmethyl, 4-nitrophenylmethyl, or4-aminophenylmethyl and R₃ is hydrogen, methyl, isopropyl, isobutyl orbenzyl; and (b) R₅ is not 2-pyridyl or 2-thiazolyl when R₁, R₃ and R₆are hydrogen, R₂ is n-pentyl and R₄ is isopropyl; and (c) R₅ and R₆ arenot both ethyl when R₁ and R₃ are hydrogen, R₂ is n-pentyl and R₄ ismethyl or isopropyl.
 2. The method of claim 1 wherein R₁ representshydrogen, or C₁-C₆ alkyl; R₃ represents hydrogen or C₁-C₆ alkyl; R₄represents the side chain of a natural or non-natural alpha amino acid;R₅ and R₆ independently represent hydrogen, C₁C₆ alkyl, or cycloalkyl orR₅ and R₆ taken together with the nitrogen atom to which they areattached form an optionally substituted saturated heterocyclic ring of 3to 8 atoms; and R₇ represents hydrogen or an acyl group.
 3. The methodof claim 1 wherein R₁ is hydrogen or methyl.
 4. The method of claim 1wherein R₂ is: C₁C₆ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl; phenyl (C₁-C₆alkyl)-, phenyl (C₃-C₆ alkenyl)-or phenyl (C₃-C₆ alkynyl)-optionallysubstituted in the phenyl ring; cycloalkyl (C₁-C₆ alkyl)-, cycloalkyl(C₃-C₆ alkenyl)- or cycloalkyl (C₃-C₆ alkynyl)-optionally substituted inthe cycloalkyl ring; heterocyclyl (C₁-C₆ alkyl)-, heterocyclyl (C₃-C₆alkenyl)- or heterocyclyl (C₃-C₆ alkynyl)-optionally substituted in theheterocyclyl ring.
 5. The method of claim 1 wherein R₂ is methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, 2-methoxyethyl, prop-2-yn-1-yl,3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl) prop-2-yn-1-yl,but-2-yn-1-yl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl,phenylpropyl, 4-chlorophenylpropyl, 4-methylphenylpropyl, or4-methoxyphenylpropyl.
 6. The method of claim 1 wherein R₂ is n-butyl,benzyl or cyclopentylmethyl.
 7. The method of claim 1 wherein R₃ ishydrogen or methyl.
 8. The method of claim 1 wherein R₄ is: a group-(Alk)_(n)R₉ where Alk is a C₁-C₆ alkyl or C₂-C₆ alkenyl group optionallyinterrupted by one or more —O—, or —S—atoms or —N(R₁₂)- groups where R₁₂is a hydrogen atom or a C₁-C₆ alkyl group, n is 0 or 1, and R₉ is anoptionally substituted cycloalkyl or cycloalkenyl group; or a benzylgroup substituted in the phenyl ring by a group of formula OCH₂COR₈where R₈ is hydroxyl, amino, C₁-C₆ alkoxy, phenyl (C₁-C₆) alkoxy, C₁-C₆alkylamino, di(C₁-C₆ alkyl)amino, (phenyl (C₁-C₆ alkyl))amino, or aheterocyclic (C₁-C₆) alkyl group, either being unsubstituted or mono-ordisubstituted in the heterocyclic ring with halo, nitro, carboxy, C₁-C₆alkoxy, cyano, C₁-C₆ alkanoyl, trifluoromethyl, C₁-C₆ alkyl, hydroxy,formyl, amino, C₁-C₆ alkylamino, di-(C₁-C₆ alkyl)amino, mercapto, C₁-C₆alkylthio, hydroxy(C₁-C₆ alkyl), mercapto(C₁-C₆ alkyl), or (C₁-C₆)alkylphenylmethyl; or a group -CR_(a)R_(b)R_(c) in which: each of R_(a),R_(b) and R_(c) is independently hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, phenyl (C₁-C₆) alkyl, (C₃-C₈) cycloalkyl; or R_(c) ishydrogen and R_(a) and R_(b) are independently phenyl or heteroaryl; orR_(c) is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, phenyl(C₁-C₆ alkyl), or C₃-C₈ cycloalkyl, and R_(a) and R_(b) together withthe carbon atom to which they are attached form a 3 to 8 memberedcycloalkyl or a 5-to 6-membered heterocyclic ring; or R_(a), R_(b) andR_(c) together with the carbon atom to which they are attached form atricyclic ring; or R_(a) and R_(b) are each independently C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, phenyl (C₁-C₆ alkyl), or a group asdefined for R_(c) below other than hydrogen, or R_(a) and R_(b) togetherwith the carbon atom to which they are attached form a cycloalkyl orheterocyclic ring, and R_(c) is hydrogen, —OH, —SH, halogen, —CN, —CO₂H,C₁-C₄ perfluoroalkyl, —CH₂OH, —CO₂(C₁-C₆alkyl), —O(C₁-C₆ alkyl), —O(C₂-C₆ alkenyl), —S(C₁-C₆ alkyl), SO(C₁-C₆ alkyl), SO₂(C₁-C₆ alkyl),—S(C₂-C₆ alkenyl), —SO(C₂-C₆ alkenyl), —SO₂(C₂-C₆ alkenyl) or agroup-Q-W wherein Q represents a bond or —O—, —S—, —SO— or —SO₂—and Wrepresents a phenyl, phenylalkyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkylalkyl, C₄-C₈ cycloalkenyl, C₄-C₈ cycloalkenylalkyl, heteroarylor heteroarylalkyl group, which group W may optionally be substituted byone or more substituents independently selected from, hydroxyl, halogen,—CN, —CO₂H, —CO₂(C₁-C₆alkyl), —CONH₂, —CONH(C₁-C₆alkyl), —CONH(C₁-C₆alkyl)₂, —CHO, —CH₂OH, C₁-C₄ perfluoroalkyl, —O(C₁-C₆ alkyl), —S(C₁-C₆alkyl), —SO(C₁-C₆) alkyl, —SO₂(C₁-C₆ alkyl), —NO₂, —NH₂,—NH(C₁-C₆)alkyl, —N(C₁-C₆ alkyl)₂, —NHCO(C₁-C₆) alkyl), C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₄-C₈cycloalkenyl,phenyl or benzyl.
 9. The method of claim 1 wherein R₄ is phenyl,tert-butyl, isobutyl, benzyl, cyclohexylmethyl, pyridin-3-ylmethyl,tert-butoxymethyl, tert-butyl, 1-benzylthio- 1-methylethyl,1-methylthio-1-methylethyl, or 1-mercapto- 1-methylethyl.
 10. The methodof claim 1 wherein R₄ is tert-butyl.
 11. The method of claim 1 whereinR₅ and R₆ are independently hydrogen, methyl, ethyl or cyclohexyl. 12.The method of claim 1 wherein R₅ and R₆ when taken together with thenitrogen atom to which they are attached form a saturated 5-to 8memberedmonocyclic N-heterocyclic ring which is attached via the N atom andwhich optionally contains NR₁₁ wherein R₁₁ is hydrogen, C₁-C₆ alkyl,benzyl, acyl, or an amino protecting group, O, S, SO or SO₂ as a ringmember, and is optionally substituted on one or more C atoms by hydroxy,C₁-C₆ alkyl, C₁-C₆ alkoxy, oxo, ketalised oxo, amino, mono(C₁-C₆alkyl)amino, di(C₁-C₆ alkyl)amino, carboxy, C₁-C₆ alkoxycarbonyl,hydroxymethyl, C₁-C₆ alkoxymethyl, carbamoyl, mono(C₁-C₆ alkyl)carbamoyl, di (C₁-C₆ alkyl) carbamoyl, or hydroxyimino.
 13. The methodof claim 1 wherein R₅ and R₆ when taken together with the nitrogen atomto which they are attached form a substituted or unsubstituted1-pyrrolidinyl, piperidin-1-yl, 1-piperazinyl, hexahydro-1-pyridazinyl,morpholin-4-yl, tetrahydro-1,4-thiazin-4-yl, tetrahydro-1,4-thiazin-4-yl1-oxide, tetrahydro-1,4-thiazin-4-yl 1,1-dioxide, thiazolidin-3-yl,hexahydroazipino, or octahydroazocino ring.
 14. The method of claim 1wherein R₇ is hydrogen, or a group R₂₀C(O)- where R₂₀ is a C₁-C₆ alkylgroup.
 15. The method of claim 1 wherein R₇ is a group R₂₀C(O)- whereR₂₀ is methyl or ethyl.
 16. The method of claim 1 wherein the compoundis N¹-(1S- dimethylcarbamoyl-2,2-dimethyl-1-propyl)—N⁴-hydroxy-2R-butyl-succinamide or a pharmaceuticallyacceptable salt, hydrate or solvate thereof.